NONLINEAR DECOMPOSITION OF HEME UV-VIS SPECTRA

我们建议扩展和联合收割机的一系列分析工具， 用于解释和分析紫外-可见光谱的典型 化学和生物化学系统。 数字化的广泛使用，快速- 研究人员对广泛的 问题，创造了发展和可用性的需要， 这些技术。 这一发展将通过研究实现， 一系列越来越复杂的血红素（铁卟啉）配体 均衡 第一个要研究的体系是 mesotetraphenylporphyrinatoiron（II），Fe（II）TPP，与1，2-二甲基咪唑， 1，2-溴。 由于这种碱只结合一次-在中等浓度下-它 滴定由两种组分组成; Fe（II）TPP和Fe（II）TPP（1，2-dichloro）。 的 两个相应的纯紫外-可见光谱很容易获得，因此标准 方法将用于重现结合常数。 光谱 该滴定曲线将进一步处理如下：1） 因子分析分解（FA），以及2）拟合一系列 高斯/洛伦兹函数（GLF）。 使用这些分析的目的 是为了确定每一个可以复制的准确性; a）绑定 常数，和B）单个纯光谱。 除了有 这两种分析中每一种的统计方法， 技术使用非线性最小二乘曲线拟合过程。 的 马夸特算法是一种非常有效的方法，但可能会错过 全局最小值如果最初的猜测是关闭的。 改进的方法 将探索参数空间的搜索。 在对两组分系统进行研究之后，FA和GLF技术将 可以扩展到由三个、四个、六个和九个组成的系统 件. 这些系统将由各种组合的 铁卟啉Fe（II）TPP和meso-tera（α，α-o- 新戊酰氨基卟啉铁（II）、铁（II）（TpivPP）、“栅栏”血红素，以及 碱1-甲基咪唑、1，2-二甲基咪唑、1，5- 二环己基咪唑、吡啶和4-苯基吡啶。 由于几个 纯光谱和结合常数是已知的，或者对它们的估计是 可用，拟议分析的开发和准确性将 每一步都经过严格的测试和评估。 待使用的配体是 除了一种以外所有的卟啉都是市售的， “栅栏”卟啉，其合成和纯化是一系列的 2个高产率步骤：1）缩合meso-tera-α， α-O-氨基苯基卟啉，其分离自市售的 通过柱色谱法可获得阻转异构体的混合物。 2)插入 通过将FeBr加入到该卟啉中， THF/CHCl 3。 将为主机和微型计算机开发软件。 的 初步的时间表是建立实验室， 第一年的系统。 扩展到更复杂的 系统将占用第二年和第三年的大部分时间。 以下 几年后，将看到软件应用到不同的系统， 生化兴趣

We propose to extend and combine a series of analytical tools for general use in the interpretation and analysis of UV-visible spectra of typical chemical and biochemical systems. The widespread use of digitizing, fast- scanning spectrophotometers by investigators studying a wide range of problems, has created the need for the development and availability of these techniques. This development will be brought about through the study of a series of progressively more complicated heme (iron porphyrin)-ligand equilibria. The first system to be studied is the titration of mesotetraphenylporphyrinatoiron(II), Fe(II)TPP, with 1,2-dimethylimidazole, 1,2-DMI. Since this base only binds once-at moderate concentrations-its titration consist of two components; Fe(II)TPP and Fe(II)TPP(1,2-DMI). The two corresponding pure UV-vis spectra are easily obtainable and so standard methodologies will be used to reproduce the binding constant. The spectral curves of this titration will then be further treated as follows: 1) Factor Analytical decomposition (FA), and 2) Fitting to a series of Gaussian/Lorentzian functions (GLF). The goal behind using these analysis is to determine the accuracy with which each can reproduce; a) the binding constant, and b) the individual pure spectra. In addition to the statistical methods of each of these two analysis, each of these two techniques uses nonlinear least-squares curve fitting procedures. The Marquardt algorithm is a very efficient method of doing this, but may miss global minima if the initial guesses are off. Methods for improved searching of parameter space will be explored. Following work on the two component system the FA and GLF techniques will be extended to systems composed of three, four, six and finally nine components. These systems will be composed of various combination of the iron porphyrins Fe(II)TPP and meso-tera(alpha, alpha, alpha, alpha-o- pivalamidoporphyrinatoiron(II), iron(II)(TpivPP), "picket fence" heme, and the bases 1-Methylimidazole, 1,2-dimethylimidazole, 1,5- dicyclohexylimidazole, pyridine and 4-phenylpyridine. Since several of the pure spectra and binding constants are known, or estimates of them are available, the development and accuracy of the proposed analyses will be tested, and critically evaluated, at each step. The ligands to be used are all commercially available as are the porphyrins with the exception of one, "picket fence" porphyrin, whose synthesis and purification is a series of 2 high yield steps: 1) condensation of meso-tera-alpha, alpha, alpha, alpha-o-aminopnenylporphyrin which is isolated form the commercially available mixture of atropisomers via column chromatography. 2) Insertion of iron to this porphyrin by adding FeBr to this porphyrin in refluxing THF/CHCl3. Software will be developed for both mainframe and microcomputers. The tentative timetable is to set up the lab and characterize the two and three component systems in the first year. Extensions to the more complicated systems will take up the second year and most of the third. Following years will see applications of the software to different systems of biochemical interest.